21 EFFECT OF SEVERAL PARAMETERS ON PARTHENOGENETIC BOVINE EMBRYO DEVELOPMENT IN VITRO

Abstract

The activation of oocytes is one of the most important steps for a successful cloning and has great importance on embryo development in vitro. The objective of this study was to examine the different parameters affecting parthenogenetic embryo development in vitro. In the first experiment, two activation protocols were compared to examine the effect of electrical pulse on activation. Bovine oocytes isolated from slaughterhouse ovaries were matured in TCM-199 supplemented with fetal bovine serum (FBS), sodium pyruvate, penicillin/streptomycin, rat insulin-like growth factor (rIGF-1), bovine follicle-stimulating hormone (bFSH), and bovine luteinizing hormone (bLH). A group of oocytes was exposed to a DC pulse of 133 V/500 µm for 25 µs, and then activated by calcium ionophore (5 µM) for 10 min, cytochalasin D (CD) (2.5 µg/mL) + cycloheximide (CHX, 10 µg/mL) for 1 h, and CHX alone for 5 h (Group 1). Another group of oocytes was activated only by chemicals without electrical pulse. Activated oocytes were cultured for 72 h in G1-3 and then 4-6 days in G2-3 medium. In the second experiment, oocytes activated by electrical pulse and chemicals were cultured in Barc medium for 7-9 days or 72 h in G1-3 and then 4-6 days in G2-3 medium. In the third experiment, oocytes activated by electrical pulse and chemicals were cultured for 48 h or 72 h in G1-3 and then 5-7 days or 4-6 days in G2-3 medium. The differences among groups were analyzed by one-way ANOVA after arcsin square transformation. In the first experiment, cleavage rate (75.6%), development rate (37.3%), and blastocyst cell number (78.4 ± 3.2) of oocytes activated by electrical pulse was higher than for the group without electrical pulse (28.7%, 8.0%, 59.5 ± 4.3, respectively; P < 0.05). This result showed that activation was started more effectively by electrical pulse than by chemicals. In the second experiment, there was no significant difference on cleavage rate between the two groups (66.6%, 65.0%, respectively), and the blastocyst development rate of parthenogenetic embryos cultured in G1-3/G2-3 (36.6%) was higher than in the Barc medium group (16.6%; P < 0.05). This result showed that G1-3/G2-3 medium was more effective for parthenogenetic embryo development than Barc medium. In the third experiment, although significant differences could not be found between the two groups in the development rate of parthenogenetic embryos cultured for a total of 7-9 days (30.8%, 39.2%, respectively), the development rate of embryos cultured for 72 h in G1-3 was higher (26.4%) than for the 48-h group (15%; P < 0.05) on Day 7. This result showed that embryos developed more slowly when cultured for a shorter time in G1-3 medium before transfer to G2-3 medium.